1. Field of the Invention
The neuropeptide bombesin exerts physiological effects by binding to specific receptors present on cells in the gastrointestinal tract and central nervous systems. Bombesin also binds to, and promotes the growth of, some types of tumor cells. The invention relates to novel bombesin derivatives which act as antagonists of bombesin, or related peptides such as gastrin releasing peptide, by blocking the binding of such peptides to their cognate receptors. By blocking the binding of bombesin-like peptides to their receptors, these antagonists block the physiological effects of these peptides and inhibit the growth of tumor cells that respond to the growth-promoting action of bombesin. Thus, these antagonists have therapeutic use in the treatment or prevention of some types of cancer, in controlling physiological effects in gastrointestinal disorders such as peptic ulcers or pancreatitis, and in modulating responses of the central nervous system including those that occur in feeding disorders or hypothermia.
2. Brief Description of the Background Art
Bombesin (hereinafter referred to as BBN) was discovered in 1970 as a potent smooth muscle contracting agent of nonmammalian origin first isolated from amphibian skin (Erspamer et al., J. Pharm. Pharmacol. 22:275 (1970)). Immunolocalization has indicated the presence of both BBN-like peptides and BBN receptors in the mammalian central nervous system and gastrointestinal tract. (Moody et al., Proc. Natl. Acad. Sci. USA 75:5372 (1978)). BBN-like peptides function in feeding and satiety and stimulate gastrin release and pancreatic exocrine secretion (Gibbs et al., Nature 282:208 (1979)); Bertaccini et al.,Br. J. Pharmac. 59:219 (1974)). As determined by immunolocalization, BBN-like peptides are present in moderate amounts in discrete areas of the brain such as the lateral hypothalamus, which controls feeding behavior and body temperature (Crawley, J., Ann. N.Y. Acad. Sci. 448:1 (1985)). BBN also potently produces hypothermia in cold-adapted rats upon central nervous systems (CNS) and peripheral administration. The first significant structure-activity studies of BBN conducted with synthesized analogs measured hypothermia and brain receptor binding as indicators of CNS activity. (Rivier and Brown, Biochem. 17:1766 (1978); Marki et al., Peptides 2:169 (1981); Moody et al., Proc. Natl. Acad. Sci. USA 75:5372 (1978)).
The sequence of BBN has been determined and is (SEQ ID No.:1) ##STR1## wherein pGlu indicates pyroglummic acid and Met-NH.sub.2 indicates methionine amide. (Erspamer, V., Ann. New York Acad. Sci. 547:3 (1988)).
Mammalian proteins related to BBN are known and include gastrin releasing peptide (hereinafter referred to as GRP) and neuromedins B and C. GRP is a 27 residue peptide that has a sequence (i.e., Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-Pro-Arg-Gly-As n-His-Gly-Tep-Ala-Val-Gly-His-Leu-Met-NH.sub.2 ; SEQ ID No.:15) that includes a carboxyl terminal sequence that is clearly related to the sequence of BBN (MacDonald et al., Biochem Biophys. Res. Commun. 90:227 (1979)). As predicted by the effects of BBN on mammalian central nervous system activities, such as the regulation of cardiovascular functions (Fisher et al., Amer. J. Physiol.: Heart and Circulatory Physiol. 17:H425 (1985)), and the immunoreactivity of antibodies to BBN and GRP to tissues of the brain and spinal cord (Panula et al., Ann. New York Acad. Sci. 547:54 (1988), BBN/GRP-like peptides are found in the CNS. Neuromedins B and C were originally isolated from the porcine CNS. Neuromedin B is a 32 residue peptide that has a sequence (Ala-Pro-Leu-Ser-Trp-Asp-Leu-Pro-Glu-Pro-Arg-Ser-Arg-Ala-Ser-Lys-Ile-Arg-V al-His-Ser-Arg-Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH.sub.2 ; SEQ ID No.:16) that includes a carboxyl terminal sequence that is clearly related to the carboxyl termini of BBN and GRP, and neuromedin C is a decapeptide that is identical to the carboxyl-terminus of GRP (i.e., Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 ; SEQ ID No.:2) (Minamino et al., Biophys. Res. Comm. 114:541 (1983); Minamino et al., Biochem. Biophys. Res. Comm. 119:14 (1984)).
Although the term "bombesin receptors" is used and accepted in the art, bombesin per se does not exist in mammalian systems. That is, the in vivo role of bombesin receptors is not to bind BBN, an amphibian peptide; rather, when used in regard to mammalian systems, the term "bombesin receptors" refers to a class of cellular receptors that bind GRP, neuromedin B or C, or other BBN-related peptides endogenous to mammals. Because BBN is closely related to GRP and similar peptides, these receptors also bind BBN. The fact the BBN not only binds to receptors on mammalian cells, but also stimulates the same intracellular effects as GRP, further indicates the close relatedness of BBN to GRP. This high degree of relatedness indicates that changes in the peptide sequences of BBN or GRP that substitute an amino acid found in one peptide for the amino acid found in the corresponding position in the other peptide will not produce functional differences. Moreover, chemical modifications that have a desirable effect on one member of the BBN/GRP-like family of peptides are expected to be readily applicable to any other member.
Nomenclature of the BBN/GRP-like family of peptides is complicated by the fact that the peptides are synthesized in vivo with amino-terminal signal peptides and multiple forms of the GRP precursor, containing differing carboxyl-terminal extension peptides (CTEPs), are known to be expressed in various cell lines (Hamad et al., Virchows Arch. 411:185 ( 1987); MacDonald et al., Biochem Biophys. Res. Commun. 90:227 (1979)). Ultimately, a BBN/GRP precursor molecule is processed to release the amino-terminal signal peptide and the CTEP, and the BBN/GRP-like peptide itself. Interestingly, both mammalian and amphibian members of the BBN/GRP family share these complex regulatory features (Spindel et al., Ann. New York Acad. Sci. 547:10 (1988)). In any event, a system of nomenclature for the BBN/GRP-like family of peptides has been proposed (Erspamer et al., Ann. New York Acad. Sci. 547:1 (1988)) and is followed herein.
BBN is one of the most powerful growth factors capable of acting alone to stimulate the mitotic growth and proliferation of cells. (Rozengurt, E., Science 234:161 (1986)). Because BBN is a highly effective mitogen, inhibitors of BBN can prevent growth and proliferation of cells, as has been shown for Swiss 3T3 fibroblasts, small cell lung carcinoma (SCLC) and neuroblastoma cells. Early events in the induction of mitotic growth of Swiss 3T3 fibroblast cells, as well as other embryonic and cancer cells, have been characterized. Biochemical indicators of early events in the mitogenic response include increases in intracellular Ca.sup.++ levels, protein kinase C activation, and thymidine uptake. The initial event in induction of mitogenic growth, binding of a BBN/GRP-like peptide to a cellular receptor, is also a biochemical event that can be monitored with appropriate assays.
The current state of the art shows a role for peptide receptors in carcinogenesis and tumor metastases. Receptors for GRP and the role of BBN/GRP-like peptides as autocrine growth factors for these cells have been described (Moody and Pert, Biochem Biophys. Res. Commun. 90:7 (1979)). High levels of BBN/GRP-like peptides and their receptors have been found in SCLC cells (Pullan et al., Br. Med. J. 1:758 (1980)). Cellular control through peptide receptors has also been demonstrated for breast and prostate gland tumors, and pancreatic carcinomas which can be treated with somatostatin analogues (Redding and Schally, Proc. Natl. Acad. Sci. USA 81:248 (1984); Redding and Schally, Proc. Natl. Acad. Sci. USA 78:6509 (1981); Hierowski et al., F.E.B.S. Letters 179:252 (1985)). In addition, malignant cells are known to be chemotactic for certain peptides. In particular, SCLC cells are known to have high-affinity receptors for BBN, GRP and related peptides (Moody et al., Life Sci. 37:105 (1985)).
Because of the role in cellular receptors in inducing the mitotic growth of cells, it was thought that substances that prevent the interaction of BBN/GRP-like peptides with their receptors might have an anti-mitotic effect upon BBN- and GRP-responsive tumor cell lines.
SCLC, which accounts for 20% of lung tumors, often includes cell lines that secrete GRP and demonstrate a mitogenic response when treated with BBN (Thomas et al., Cancer Res. 52:4872 (1992)). SCLC cell lines may be isolated from tumors and tested in vitro for their response to potential bomesin receptor antagonists (Trepel et al., Biochem. Biophys. Res. Commun. 156:1383 (1988); Mahmoud et al., Life Sci. 44:367 (1989)). Cultured SCLC cells, or biopsy samples containing SCLC cells, produce tumors when subcutaneously introduced into athymic nude mice (Brambilla et al., Cancer (Phila.) 64:1238 (1989)). The potency of antitumor agents may be examined in such mice, thus allowing potential anticancer agents to be evaluated in a live animal model (Cuttitta et al., Nature 316: 823 (1985)). Thus, SCLC is a useful system for developing antitumor agents to treat BBN/GRP-responsive cancers.
A monoclonai antibody that binds the carboxy-terminal portions of BBN and GRP, and which prevents the binding of GRP to its receptors, inhibits the in vitro and in vivo growth of SCLC cells (Cuttitta et al., Nature 316: 823 (1985)). The clinical use of this monoclonal antibody is being investigated (Mulshine et al., Ann. New York Acad. Sci. 547:360 (1988)).
Recently, attempts have been made to develop BBN/GRP antagonists that bind to cellular receptors by synthesizing chemical structures that are derived from the structures of BBN or GRP. Such receptor antagonists would inhibit the physiological effects stimulated by endogenous BBN/GRP-like peptides by limiting the ability of cells to bind these peptides.
Chemical structures derived from BBN include derivatives of BBN which retain 14 amino acid residues but in which dextrorotatory aromatic residues have been substituted for His.sup.12 (Coy et al., Ann. New York Acad. Sci. 547:150 (1988); Saeed et al., Peptides 10:597 (1989)), derivatives of the carboxyl terminal 7-14 octapeptide in which Leu replaces Met.sup.14 and individual peptide bonds are reduced (Coy et al., J. Biol. Chem. 263:5056 (1988); Coy et al., J. Biol. Chem. 264:14691 (1989)) or in which N-isobutyryl-His replaces Gln.sup.7 and dextrorotatory Alanine replaces Gly.sup.11 (Camble et al., Life Sci. 45:1521 (1989)), and derivatives of the carboxyl terminal 6-13 octapeptide in which D-Phe replaces Phe.sup.6 and in which the absent Met.sup.14 residue is replaced by a series of des-Met.sup.14 alkylamides and esters (Staley et al., Peptides 12:145 (1991); Wang et al., J. Biol. Chem. 265:15695 (1990)). Other BBN analogs, as well as GRP analogs, have been described (Coy et al., J. Biol. Chem. 264:14691 (1989); Cai et al., U.S. Pat. No. 5,244,883; Kull et al., WO 92/02545; Heimbrook et al., J. Biol. Chem. 264:11258 (1989); Heimbrook et al., U.S. Pat. No. 4,943,561; Riemen et al., U.S. Pat. No. 5,019,647; Mukai et al., Peptides 11:173 (1990); Leban et al., Proc. Natl. Acad. Sci. USA 90:1922 (1993)); See also Camble et al., Kim et al., Mokotoff et al. and Castiglione et al. in Peptides: Chemistry, Structure and Biology, Rivier and Marshall, eds., ESCOM, Leiden, p. 174, p. 182, p. 63 and p. 168, respectively (1990)).
Despite the fact that a number of derivatives of BBN have been prepared, a need still exists for effective bombesin receptor antagonists that possess adequate in vivo stability for therapeutic purposes. Compounds which act as BBN/GRP antagonists in one system may also act as partial agonists in other systems (Coy et al., Eur. J. Phramacol. 190:31 (1990)). Moreover, some BBN/GRP antagonists are reversible. For example, a reduced peptide bond antagonist ([Psi.sup.13,14, Leu.sup.14 ]BBN) inhibits growth with moderate potency (i.e., K.sub.i =30 nM), but the effects of this antagonist are clearly reversed in a few minutes by the addition of BBN (Coy et at., J. Biol. Chem. 263:5056 (1988); Mahoud et al., Cancer Res. 51:1798 (1991)). Furthermore, some inhibitory analogues that have a potency in vitro that is similar to that of [Psi.sup.13,14, Leu.sup.14 ]BBN have been found, when tested, to be less potent than this antagonist in inhibiting SCLC growth in cultures and in vivo. This loss of potency may be due to susceptibility of the compound to proteolytic degradation. Some inhibitors are equipotent to GRP in binding assays and inhibit GRP-stimulated effects in human SCLC cells in vitro and in murine tissues in vivo, yet they do not actually inhibit the growth of SCLC cells in vitro (Heimbrook et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshall, eds., ESCOM, Leiden, p. 185 (1990)). Therefore, a need exists for the development of other potent BBN antagonists that do not have agonist activity, that are resistant to proteolysis and not easily reversible, and are thus more effective for a longer period of time in vivo.
Peptide hormone receptor antagonists are a current approach to new anti-tumor chemotherapeutic drugs. Since tumors are derived from endocrine cell types, they have cell surface peptide receptors, secrete peptide hormones and display a growth response to peptides (Moody et al., Science 214:1246 (1981)). Certain peptides, such as BBN and GRP, function as autocrine growth factors for such tumors (Sporn and Roberts, Nature 313:745 (1985); Zachary and Rozengurt, Proc. Natl. Acad. Sci. USA 82:7616 (1985)). One example of a tumor cell line which exhibits growth in response to a peptide is human SCLC, which demonstrates a mitogenic response to GRP, the mammalian homologue of BBN ((Moody et al., Science 214:1246 (1981); Sporn and Roberts, Nature 313:745 (1985); Carney et al., Clin. Res. 31:404A (1983); Cuttitta et al., Nature 316:823 (1985)). It is now known that SCLC is a transformed neuroendocrine cell type which secretes, and has receptors for, GRP. Some recently reported bombesin receptor antagonists are known to inhibit growth of SCLC cells in vitro, yet do not demonstrate the same efficacy when administered in vivo (Mahoud et al., Cancer Res. 51 (1798-1802)). Therefore, a need also exists for the development of bombesin antagonists containing structural modifications which enhance stability and specificity for in vivo use.